1. Field of the Invention
The present invention relates to an avian polyomavirus vaccine and to a method of preventing avian polyomavirus infection in Psittaciformes. 
2. Background Art
The first acute, generalized infection associated with avian polyomavirus was described in 1980 in young psittacine birds and was called budgerigar fledgling disease (Davis, R. R. et al., xe2x80x9cA viral disease of fledgling budgerigars,xe2x80x9d Avian Dis., 1981, 25:179-183; Bozeman, L. H., et al., xe2x80x9cCharacterization of a papovavirus isolated from fledgling budgerigars,xe2x80x9d Avian Dis., 1981, 25:972-980; Bernier, G., et al., xe2x80x9cA generalized inclusion body disease in the budgerigar (Melopsittacus undulatus) caused by a papovavirus-like agent,xe2x80x9d Avian Dis., 1981, 25:1083-1092; Dykstra, M. J., et al., xe2x80x9cInvestigations of budgerigar fledgling disease virus,xe2x80x9d Am. J. Vet. Res., 1984, 45:1883-1887; Lehn, H., Muller, H., xe2x80x9cCloning and characterization of budgerigar fledgling disease virus (BFDV), an avian polyomavirus,xe2x80x9d Virology, 1986, 151:362-370). Since its discovery in 1980, avian polyomavirus has been associated with disease in a number of different species of companion and aviary birds including Budgerigars, caiques, macaws, Amazon parrots, conures, cockatoos, lovebirds, Splendid Parakeet, Pionus Parrots, African Grey Parrots, Eclectus Parrots, Cockatiels, finches and lories (Davies et al., 1981; Bozeman et al., 1981; Bernier et al., 1981; Lehn and Muller, 1986; Jacobson, E. R., et al., xe2x80x9cEpornitic of papova-like virus-associated disease in a psittacine nursery,xe2x80x9d J. Am. Vet. Med. Assoc., 1984, 185:1337-1341; Clubb, S. L., Davis, R. B., xe2x80x9cOutbreak of papova-like viral infection in a psittacine nursery-a retrospective view,xe2x80x9d Proc. Assoc. Avian Vet., Toronto, 1984, 121-129; Graham, D. L., xe2x80x9cAn update on selected pet bird virus infections,xe2x80x9d Proc. Assoc. Avian Vet., Toronto, 1984, 267-280; Gaskin, J. M., xe2x80x9cPsittacine viral disease: A perspective,xe2x80x9d J. Zoo. Wildl. Med., 1989, 20:240-264; Johnston, K. M., Riddell, C., xe2x80x9cIntranuclear inclusion bodies in finches,xe2x80x9d Can. Vet. J., 1986, 27:432-434; Marshall, R., xe2x80x9cPapova-like virus in a finch aviary, Proc. Assoc. Avian Vet., 1989, 203-207; Schmidt, R. E., et al., xe2x80x9cMorphologic identification of papovavirus in a Moluccan cockatoo (Cacatua moluceensis) with neurologic signs,xe2x80x9d Assoc. Avian Vet. Today, 1987, 1:107-108; Pass, D. A., et al., xe2x80x9cA papova-like virus infection of splendid parakeets (Neophema splendida),xe2x80x9d Avian Dis., 1987, 31:680-684; Pass, D. A., xe2x80x9cA papova-like virus infection of lovebirds (Agapornis sp.(,xe2x80x9d Aus. Vet. J.; 1985, 82:318-319).
The type of clinical disease in Budgerigars, for example, depends upon the age and condition of birds when exposure to the virus occurs. Neonates from infected flocks may develop normally for 10-15 days and then suddenly die with no premonitory signs. Other infected hatchlings may develop clinical signs that include abdominal distention, subcutaneous hemorrhage, tremors of the head and neck, ataxia and reduced formation of down and contour feathers feather abnormalities,xe2x80x9d J. Vet. Sci. 46:577-587, 1984; Bernier et al., 1984; Clubb and Davis, 1984; Schmidt et al., 1987; Histopathology Reports #SC90-0637 and #SC90-0638, Schubot Exotic Bird health Center, Texas AandM University; Vernot, J., personal communication; Dykstra, M. J., Bozeman, L. H., xe2x80x9cA light and electron microscopic examination of budgerigar fledgling disease virus in tissue and in cell culture. Avian Pathol. 11:11-18, 1982). Infections have also been associated with decreased hatchability and embryonic death (Hudson, L., Hay, F. C., xe2x80x9cIsolation and structure of immunoglobulins,xe2x80x9d Hudson, L., Hay, F. C. Ed., Practical Immunology, Boston, 1980, 156-202). Mortality rates can be as high as 100% in affected hatchlings. Surviving birds often exhibit dystrophic primary tail feathers, lack of down feathers on the back and abdomen, and lock of filoplumes on the head and neck. Additionally, surviving birds with primary feather abnormalities are usually unable to fly.
In larger psittacine birds, polyomavirus infections may cause peracute death with no premonitory signs, or acute death after development of clinical changes including depression, anorexia, weight loss, delayed crop-emptying, regurgitation, diarrhea, dehydration, subcutaneous hemorrhages, dyspnea, polyuria, and posterior paresis and paralysis (Pass et al., 1987; Johnston and Riddell, 1986; Mathey, W. J., Cho, B. R., xe2x80x9cTremors of nestling budgerigars with budgerigar fledgling disease,xe2x80x9d Proc. 33rd West Poult. Dis. Conf., 1984, 102; Woods, L., xe2x80x9cPapova-like virus in a purple finch,xe2x80x9d J. Zoo. Wildl. Med., 1989, 218-218; Gaskin, J. M., xe2x80x9cThe serodiagnosis of psittacine viral infections,xe2x80x9d Assoc. Avian Vet. Honolulu, 1988, 7-10). Characteristic lesions associated with a polyomavirus infection have been demonstrated in companion birds from the United States (Jacobson et al., 1984; Clubb and Davis, 1984; Graham, 1984), Canada (Gough, J. F., xe2x80x9cOutbreaks of budgerigar fledgling disease in three aviaries in Ontario,: Can. Vet. J., 1989, 30:672-674, Bernier et al., 1984), Japan (Hirai et al., 1984), Italy (Pascucci, S., et al., xe2x80x9cMalattia da virus papova-simile nel papagallino ondulato (Melopsittacus undulatus), Clin. Med. (Milan), 1983, 106:38-41), Hungary (Szotjkov, V., et al., xe2x80x9cA hullamous papagaj (Melopsittacus Undulatus) papovavirus okozta megbetegedesenek hazai megallapitasa, Magy Allatorv Lapja 1985, 40:50-63), Germany (Krautwald, M-E, Kaleta, E. F., xe2x80x9cRelationship of French moult and early virus induced mortality in nestling budgerigars,xe2x80x9d Proc. 8th Intl. Cong. World Vet. Poult. Assoc., 1985, 115) and Australia (Pass et al., 1987; Pass, 1985).
Immunodiffusion and virus neutralization techniques have been used to demonstrate anti-polyomavirus antibodies in psittacine birds (Jacobson et al., 1984; Clubb and Davis, 1984; Gaskin, 1989; Davis et al., 1981; Gaskin, 1988; Lynch, J., et al., xe2x80x9cIsolation and experimental chicken-embryo-inoculation studies with budgerigar papovavirus,xe2x80x9d Avian Dis. 1984, 28:1135-1139; Wainwright, P. O., et al., xe2x80x9cSerological evaluation of some psittaciformes for budgerigar fledgling disease virus,xe2x80x9d Avian Dis. 1987, 31:673-676). During epornitics in mixed psittacine bird collection, infected survivors and asymptomatic birds exposed to them have been shown to develop anti-polyomavirus neutralizing antibodies (Jacobson et al., 1984; Clubb and Davis, 1984; Wainwright et al., 1987). Seronegative young adult birds will seroconvert when housed adjacent to seropositive breeding adults; indicating that an antibody response does occur following natural exposure to the virus (Jacobson et al., 1984; Clubb and Davis, 1984; Wainwright et al., 1987; Davis, R. B., xe2x80x9cBudgerigar fledgling disease (BFD), 32nd West Poult. Dis. Conf., 1983, 104). However, prior to the present invention it had not been determined whether this antibody response could be induced through vaccination or whether the resulting immunologic response would be protective.
In the past, attempts at producing a vaccine against avian polyomavirus have been unsuccessful. The existence of subclinical infections and chronically infected carrier birds, coupled with a lack of understanding of the epidemiologic and pathophysiologic characteristics of infection have all contributed to the lack of success.
Consequently, avian polyomavirus infections continue to cause high levels of mortality in companion and aviary birds, resulting in pscyhological distress for clients and financial burdens for aviculturists and retail distributors despite discovery of the virus over 14 years ago. Therefore, there exists a long-felt need in the art for a safe and effective vaccine against avian polyomavirus which is cross-protective against the disease in multiple species of Psittaciformes. 
Another problem associated with vaccine failure in Psittaciformes has been the lack of a suitable adjuvant. Two killed oil-adjuvanted herpsevirus (Pacheco""s disease virus) vaccines that were conditionally licensed for use in Psittaciformes were found to cause unacceptable reactions in a number of vaccinates, particularly cockatoos (Davis et al., 1981; Bozeman et al., 1981; Bernier et al., 1981; Dykstra et al., 1984). These reactions were characterized by the formation of abscesses (subcutaneous inoculation) or muscle necrosis (IM inoculation). In some Psittaciformes, granulomatous or necrotizing lesions were not noted until several months after vaccination. In other birds, lesions were noted within several weeks of administration of a booster vaccination (Davis et al., 1981; Bozeman et al., 1981; Schmidt et al., 1987). In some cases, deaths have been associated with the use of oil-adjuvanted vaccines.
In general, the advantage of oil-adjuvanted vaccines in comparison to other immunization products is their ability to induce durable immunity when mixed with an inactivated antigen. However, the occurrence of adverse reactions in some Psittaciformes vaccinated with oil-adjuvanted vaccines created the need for an alternative adjuvant for use with inactivated antigens intended for administration in this order of birds (Davis et al., 1981; Bozeman et al., 1981; Schmidt et al., 1987). Therefore, there exists a need in the art for a suitable adjuvant for use in Psittaciformes which augments the immune response yet does not produce an adverse reaction in the vaccinate.
The present invention satisfies the long-felt need in the art for a safe and effective vaccine to protect psittacine birds against avian polyomavirus disease by providing a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier.
In one embodiment, the vaccine is derived from a strain of inactivated avian polyomavirus known as the L4 strain. In another embodiment, the invention provides a vaccine wherein immunogenic amount of the inactivated avian polyomavirus corresponds to a titer of between 104.5TCID50 and 107TCID50 for the avian polyomavirus before inactivation, but especially about 105.8TCID50.
The present invention also satisfies the need for a suitable adjuvant for use in psittacine species by providing an adjuvant, e.g., a long chain polydispersed beta- (1,4) linked mannan polymer interspersed with O-acetylated groups such as ACEMANNAN, (Carrington Laboratories, Dallas, Tx.) for use, not only in the vaccines of the present invention, but also in other psittacine vaccines.
Also provided is a composition which produces either a primary or an anamnestic response against avain polyomavirus infection in a sensitized bird which is classified as being a member of the Psittaciformes order, comprising a primary or an anamnestic response inducing amount of a recombinant protein of avian polyomavirus and a pharmaceutically acceptable carrier. In one embodiment, the composition comprises a recombinant VP1 capsid protein of avian polyomavirus.
In another embodiment, the recombinant protein is produced in the bird""s cells by the injection of plasmids encoding the recombinant proteins. In a specific embodiment, the plasmid is constructed of nucleic acid sequences encoding the agnogene, VP2, VP3, and VP1 proteins of avian polyomavirus. Alternatively, the plasmid can be constructed without the agnogene sequence. In an alternative embodiment, the plasmid will encode one or more nucleic acid sequences from the group consisting of avian polyomavirus agnogene, VP1, VP2, and VP3 proteins.
The present invention also provides a method of preventing avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier. In one embodiment, the method further comprises administering at least one booster vaccine to the bird.
Further, the invention provides a method of preventing avian polyomavirus infection in a bird from a species which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus which infects a bird from a different species of the Psittaciformes order an a pharmaceutically acceptable carrier. The vaccines and compositions provided by the invention can be utilized in the methods provided herein.
The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.
As used in the claims, xe2x80x9caxe2x80x9d can mean one or more, depending on the context of the claim.
The present invention provides a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order. One embodiment comprises an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier. Another embodiment comprises a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of a recombinant protein derived from an avian polyomavirus and a pharmaceutically acceptable carrier.
The term xe2x80x9cimmunogenic amountxe2x80x9d means an amount of an immunogen, i.e., the inactivated avian polyomavirus or a portion thereof, which is sufficient to induce an immune response in the vaccinated bird and which protects the bird against active infection with avian polyomavirus upon exposure thereto.
The terms xe2x80x9cnucleic acid vaccinexe2x80x9d, xe2x80x9cnucleic acid vaccine vectorxe2x80x9d, and xe2x80x9cnaked nucleic acid vaccinexe2x80x9d, which are used interchangeably herein, mean a vaccine delivered in the form of a non-replicating nucleic acid.
The birds which can be treated by the invention can be any of the various species of birds which are classified as being members of the Psittaciformes order. Examples of such birds include, but are not limited to, Budgerigars (Melopsittacus undulatus), caiques (e.g., Pionites leucogaster leucogaster), macaws (e.g., Ara ararauna), Amazon parrots (e.g., Amazona ochrocephala auropalliata, courses (e.g., Pyrrhara picta, Aratinga wagleri wagleri, Aratinga solstitialis, Aratinga guarouba, Artinga holochlora rubritorquis or Aratinga acuticaudata acuticaudata), cockatoos (e.g., Cacatua moluccensis, Cacatua ducorps, Cacatua sulphura, Cacatua goffini or Cacatua alba), Splendid Parakeets (Neophema splendid), Pionus Parrots (Pionus maximillani), African Grey Parrots (Psittacus erithacus erithacus), Eclectus Parrots (Electus roratus), Cockatiels (Nymphicus hollandicus) and parakeets (e.g. Psittacula krameri krameri). Specifically exemplified by the invention is a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier wherein the bird is selected from the group consisting of a macaw, an Amazon parrot, a conure, a cockatoo, a Pionus Parrot, and an African Grey Parrot.
Given the surprising fact provided by the invention that avian polyomavirus can be prevented in multiple species of Psittaciformes utilizing a single strain of avian polyomavirus, it is contemplated that the vaccines of the present invention can be constructed from any isolated strain of avian polyomavirus which infects a member of the Psittaciformes order by utilizing the methods taught herein. For example, the subject avian polyomavirus can be isolated and cultured utilizing the method taught by Bozeman et al., 1981 or by other methods known in the art. Once isolated, the virus can be purified if desired, inactivated, the vaccine prepared and the immunogenic dose optimized by the methods taught herein.
In one embodiment of the invention, the inactivated avian polyomavirus vaccine is derived from an isolated avian polyomavirus designated the xe2x80x9cL4xe2x80x9d strain. The L4 strain was isolated from an infected Budgerigar at the University of Georgia College of Veterinary Medicine in 1981 utilizing the method of Bozeman et al., 1981, and can be obtained from the Laboratory of Dr. Phil D. Lukert, College of Veterinary Medicine, University of Georgia, Athena, Ga. 30602.
One embodiment of the invention provides a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier, wherein the immunogenic amount of the inactivated avian polyomavirus corresponds to a titer of between 104.5TCID50 and 107TCID50 for the avian polyomavirus before inactivation.
In a presently preferred embodiment, the immunogenic amount of the inactivated avian polyomavirus corresponds to a titer of 105.8TCID50 for the avian polyomavirus before inactivation. As used herein, the immunogenic amount is expressed in terms of xe2x80x9cTCID50xe2x80x9d titer which is given its common meaning in the art of a tissue culture infection dose which infects 50% of the cells of a tissue culture inoculum. Thus, the immunogenic amount of any particular strain of inactivated avian polyomavirus that is utilized to prepare the vaccines of the invention is based upon the tissue culture infectivity titer for that particular strain of virus before the virus is inactivated for vaccine preparation. Also, depending upon the species, size and condition of the bird being vaccinated, the immunogenic amount can be varied by the optimization procedures taught herein or by procedures known in the art.
The vaccines of the present invention can be used either alone or in combination with a suitable adjuvant. In one embodiment the invention provides a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of an inactivated avian polyomavirus, a pharmaceutically acceptable carrier, and an adjuvant which is suitable for use in a bird which is classified as being a member of the Psittaciformes order. The term xe2x80x9csuitablexe2x80x9d is meant to include as an adjuvant, any substance which can be used in combination with the immunogen (e.g., inactivated avian polyomavirus or portion thereof) of the vaccine to augment the immune response without producing adverse side affects in the vaccinated bird. It is contemplated by the invention that the adjuvants described herein can be utilized in a vaccine against any psittacine pathogen. The adjuvants described herein can be utilized in any species which is a member of the Psittaciformes order including, but not limited to, the examples of Psittaciformes cited herein.
In one embodiment, the suitable adjuvant is a long chain polydispersed beta- (1,4) linked mannan polymer interspersed with O-acetylated groups. The presently preferred mannan polymer of the intention is ACEMANNAN. In another embodiment, the suitable adjuvant is a deproteinized highly purified cell wall extract derived from a non-pathogenic strain of Mycobacterium species. A presently preferred Mycobacterium extract is EQUIMUNE, a deprotenized highly purified cell wall extract derived from non-pathogenic strains of Mycobacterium species (Vetrepharm Research Inc., Athens, Ga.). Yet another embodiment contemplates the use of aluminum hydroxide as the adjuvant. Given the teachings and protocols provided herein for testing adjuvants, other adjuvants known in the art can be tested and utilized.
The vaccine protocol used in administer the immunogenic amount can vary depending upon the species, size and condition of the bird. The vaccine of the invention is typically administered parenterally, either subcutaneously or intramuscularly by injection. Of course, the immunogenic amount can be given in divided doses or administered at multiple sites in the bird. Booster immunizations can be given utilizing vaccines containing whole inactivated avian polyomavirus or any immunogenic portion thereof.
In one embodiment, the invention specifically provides a composition which produces an anamnestic response against avian polyomavirus infection in a sensitized bird which is classified as being a member of the Psittaciformes order, comprising an anamnestic response inducing amount of a recombinant protein of avian polyomavirus and a pharmaceutically acceptable carrier. As used herein, the term xe2x80x9canamnestic responsexe2x80x9d means a secondary (booster) immune response in a sensitized bird. By xe2x80x9csensitized birdxe2x80x9d is means a bird which has been previously been in contact with avian polyomavirus antigen either by natural exposure to the virus or by vaccination (primary immunization) with avian polyomavirus or an antigenic portion thereof.
The invention also provides the discovery that the VP1 protein of avian polyomavirus can be utilized either as the primary immunogen or as a booster to the immune response to primary vaccination against avian polyomavirus in a safe and efficient manner and with minimal stress to the vaccinated bird. In one embodiment, the invention provides a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising an immunogenic amount of a recombinant protein derived from an avian polyomavirus and a pharmaceutically acceptable carrier. In a more preferred embodiment, the invention provides the vaccine comprising an immunogenic amount of the recombinant protein derived from an avian polyomavirus, a pharmaceutically acceptable carrier and an adjuvant suitable for use in a bird which is classified as being a member of the Psittaciformes order. In another embodiment the immunogenic amount of the recombinant protein derived from an avian polyomavirus is between about 1.0 mg. and about 3 mg.
Still another embodiment of the present invention provides a composition which produces an anamnestic (secondary) response against avian polyomavirus infection in a sensitized bird which is classified as being a member of the Psittaciformes order, comprising an anamnestic response inducing amount of a recombinant avian polyovirus VP1 capsid protein and a pharmaceutically acceptable carrier.
Briefly, the recombinant VP1 protein in one embodiment was produced in E. coli by cloning the gene that codes for this protein into the pFLAG expression vector (International Biotechnologies, New Haven, Conn.). The expressed protein was partially purified by affinity chromatography using an anti-FLAG monoclonal antibody and the composition prepared by adding the protein to sterile saline (Garcia, A. P., et al., xe2x80x9cDiagnosis of polyomavirus infection in seedcrackers using DNA in situ hybridization,xe2x80x9d J. Assoc. Avian Vet., 1993; Gaskin, 1989, see also Yeung AKH, et al. Studies on the immunoproperties of recombinant VP1 from budgerigar fledgling disease virus by cloning and expressing VP1 in E. coli [Dissertation]. University of Georgia, 1993; and Rodgers, Rebecca E.d., et al., xe2x80x9cPurification of Recombinant Budgerigar Fledgling Disease Virus VP1 Capsid Protein and Its ability for In Vitro Capsid Assembly,xe2x80x9d J. Virology, Vol. 68, No. 5, pp. 3386-3390 (May 1994)).
Given the teachings provided herein one of skill in the art will realize that other recombinant proteins and polypeptide fragments from polyoma virus can be utilized as an immunogen. Such proteins, or fragments thereof, can, for example, be obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the antigenic polypeptide or fragments thereof.
Given the amino acid sequence of the avian polyomavirus antigens (see, e.g., O. Rott et al., Virology, 1988, 165: 74-86; GenBank Accession No. M20775; R. Stoll et al., J. Gen Virology, 1993, 74:229-237), one can synthesize, using standard peptide synthesis techniques, peptide fragments chosen to be homologous to immunoreactive regions of the antigen and to modify these fragments by inclusion, deletion or modification of particular amino acid residues in the derived sequences. Thus, synthesis or purification of an extremely large number of peptides derived from the antigen is possible. Such peptides can be used to immunize a member of the Psittaciformes order.
The amino acid sequences of the present polypeptides can contain an immunoreactive portion of avian polyomavirus antigen attached to sequences designed to provide for some additional property, such as solubility. The amino acid sequences of an avian polyomavirus antigen can include sequences in which one or more amino acids have been substituted with another amino acid to provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, alter enzymatic activity, or alter interactions, e.g., at the injection site or with gastric acidity if an oral administration of the vaccine is used. In any case, the peptide must possess immunogenicity.
Recombinant viral proteins or protein fragments can be tested to determine their immunogenicity by the methods taught in the examples or by other methods known in the art. Briefly, various concentrations of a putative immunogenically specific fragment are prepared and administered to a bird and the immunological response (e.g., the production of antibodies or cell mediated immunity) of the bird to each concentration is determined. The amount of antigen administered will depend upon the species, size and condition of the bird. Thereafter an animal so inoculated with the antigen can be exposed to virulent avian polyomavirus to test the potential vaccine effect of the specific immunogenic fragment. The specificity of the putative immunogenic fragment can be ascertained by testing sera, and other fluids or lymphocytes form the inoculated bird, for cross-reactivity with other closely related avian polyomaviruses. Once the immunogenicity of a viral fragment is established, the immunogenic amount to be administered to a particular bird can be determined by optimization procedures as taught herein and known in the art.
In addition to the E. coli expression vectors herein, there are numerous E. coli expression vectors known to one of ordinary skill in the art useful for the expression of antigenic avian polyomavirus proteins and polypeptide fragments. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a betalactamase promoter system, or a promoter system for phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary an amino terminal methionine can be provided by insertion of a Met codon 5xe2x80x2 and fused in-frame with the antigen. Also, the carboxy-terminal extension of the antigen can be removed using standard oligonucleotide mutagenesis procedures.
Additionally, yeast expression can be use. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion system exhibits correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader regions (encoded by the MF-alpha-1 gene) is routinely used to direct protein secretion from yeast. The leader regions of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence. The antigen coding sequence can be fused in-frame to the pre-pro-alpha-factor leader regions. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The antigen coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the antigen coding sequences can be fused to a second protein coding sequence, such as Sj26 or beta-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast.
The DNA sequences can be expressed in hosts after the sequences have been operably linked to, i.e., positioned to ensure the functioning of an expression control sequence in an appropriate expression vector. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors can contain selection makers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362).
Polynucleotides encoding a variant polypeptide may include sequences that facilitate transcription (expression sequences) and translation of the coding sequences such that the encoded polypeptide product is produced. Construction of such polynucleotides is well known in the art. For example, such polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts, and, optionally, sequences necessary for replication of a vector.
One example of a eukaryotic expression vector is the baculovirus insect vector. Expression can be achieved for example in Spodoptera frugiperda (SF9) cells using the polyhedron promoter with the target nucleic acid upstream of the promoter.
It is specifically contemplated that the avian polyomavirus expressed proteins can be used as the anitgen for ELISA testing to identify the presence of avian polyomavirus antibodies from the blood or other specimens from birds.
The vaccines and compositions of the invention can include, as noted above, an effective amount of inactivated avian polyomavirus in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. Actual methods of preparing dosage forms are known, or will be apparent, to those skilled in this art; for example, see Martin, E. W., Ed., Remington""s Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, Pa.
Parenteral administration is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795.
The present invention also provides a method of preventing avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier. The subject bird of the methods of the invention can be any of the various species of birds which are classified as being members of the Psittaciformes order including, but not limited to, the examples cited herein. Specifically provided, however, is a method of preventing avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier, wherein the bird is selected from the group consisting of a macaw, an Amazon parrot, a conure, a cockatoo, a Pionus Parrot, and an African Grey Parrot.
Also provided is a method of preventing avian polyomavirus infection in a bird from a species which is classified as being a member of the Psittaciformes order, comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus which infects a bird from a different species of the Psittaciformes order and a pharmaceutically acceptable carrier.
In one embodiment, the vaccine utilized in the methods of the invention is derived from an isolated avian polyomavirus designated the L4 strain. Given the surprisingly broad species coverage of the L4 strain vaccine as provided herein, other strains of avian polyomavirus isolated from Psittaciformes bird can be utilized to produce the vaccines of the invention and utilized in the above methods to cross-protect multiple species of Psittaciformes with a single vaccine. The L6 strain is a strain which can also be utilized.
Any of the vaccines and compositions described herein can be utilized in the methods of the invention, where appropriate, to prevent infection with or booster immunity to avian polyomavirus in a subject bird. For example, the vaccine utilized in the methods of the invention can further comprise an adjuvant suitable for use in a bird which is classified as a member of the Psittaciformes order. The adjuvant can be a long chain polydispersed xcex2 (1, 4) linked mannan polymer interspersed with O-acetylated groups such as, e.g., ACEMANNAN or a deproteinzied highly purified cell wall extract derived form a non-pathogenic strain of Mycobacterium species such as, e.g., EQUIMUNE.
In the methods described herein, the administering step is typically performed by parenteral administration, i.e., subcutaneous or intramuscular injection of the vaccine into the subject bird. The immunogenic amount of vaccine utilized in the methods of the invention is the same as the provided for in the vaccines of the invention. Specifically, the immunogenic amount of the inactivated avian polyomavirus corresponds to a titer of between 104.5TCID50 and 107TCID50 for the avian polyomavirus before inactivation but especially about 105.8TCID50.
The methods of the invention can further comprise the step of administering at least one booster vaccine to the bird. One or more booster inoculations are typically administered at bi-weekly intervals. The booster vaccine can be any of the vaccine preparations contemplated herein. However, a preferred embodiment of the invention provides a method of preventing avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order, the composition comprising administering to the bird a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier. After the initial inoculation, at least one booster vaccine is administered to the bird. The booster vaccine is a composition which produces an anamnestic response against avian polyomavirus infection in a sensitized bird which is classified as being a member of the Psittaciformes order. The booster comprises an anamnestic response inducing amount of a recombinant protein of avian polyomavirus and a pharmaceutically acceptable carrier. The booster vaccine can be comprised of any recombinant protein derived from avian polyomavirus or an immunogenic polypeptide fragment thereof. In one embodiment, the recombinant protein is the VP1 capsid protein. Briefly, the first booster vaccine can be administered to the subject bird about two weeks following primary inoculation. If desired, a second booster can be administered in about two weeks.
A recombinant protein such as the VP1 protein produces a specific antibody response in the animal to only a portion of the virus. Such a response to a specific immunogenic protein greatly reduces the risks associated with either a primary immunization or with a booster vaccination. Reaction to the recombinant vaccine is therefore milder yet sufficiently immunogenic to generate in the bird a protective immunity to the virus. In addition to being safer and less stressful, vaccines derived from recombinant proteins are more economical to manufacture.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.